Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for approximately 2 million deaths each year and remains a major public health hazard throughout the world. Existing treatments for both drug-sensitive TB and multi-drug resistant TB (MDR-TB) require prolonged treatment durations (6 months for drug-sensitive TB, 18-24 months for MDR-TB) and are difficult to administer. The inclusion of a fluoroquinoline antibiotic, which targets the A subunit of DNA gyrase (GyrA), into combination therapy regimens has improved culture conversion rates in clinical trials, indicating a potential to shorten treatment duration. Given the inevitable development of fluoroquinoline drug resistance, the B subunit of DNA gyrase (GyrB) is an attractive alternative target, with the potential to similarly shorten treatment length and perhaps also treat latent TB. The current classes of gyrase B inhibitors have only moderate anti-TB activity in vitro and in vivo, most likel due to their inability to efficiently penetrate the thick mycolic acid cell wall barrier of Mtb. Ths proposal will use a target-based screening strategy to rapidly identify new chemical scaffolds targeting GyrB with greater potential to be developed into anti-TB drugs. To accomplish this, structural information from existing high-affinity inhibitors and cheminformatics design principles will be used to design a GyrB-targeted combinatorial library to more efficiently explore the scaffold chemical space for this target. Parallel synthesis methodologies will be used to rapidly synthesize and purify this targeted library, which will then be screened for binding to gyrase B using a fluorescence polarization binding assay. New classes of inhibitors will then be profiled for microbiological activity and evaluated as new lead antitubercular compounds. This rational screening approach could potentially produce an important new class of antitubercular drugs with activity against MDR-TB and the ability to shorten treatment duration.